Use of substances for treating tumors

ABSTRACT

The present invention relates to the use of an active ingredient and to a method for the prevention or treatment of tumors, the diagnostic detection of disorders associated with these tumors, and pharmaceutical compositions and kits related thereto.

The present invention relates to the use of an active ingredient and toa method for the prevention or treatment of tumors, the diagnosticdetection of disorders associated with these tumors, and pharmaceuticalcompositions and kits related thereto.

By tumor is meant a swelling or the localized increase in tissue volume.In the wider sense it may mean any localized swelling, e.g. through anedema, an acute or chronic inflammation, aneurysmal dilatation, aninflammation-related organ swelling (e.g. a so-called splenicenlargement). In the narrower sense, tumor means tissue neoplasms(growth, blastoma, neoplasia) in the form of a spontaneous, autonomousand irreversible excessive growth, with various degrees ofdisinhibition, of endogenous tissue, which is usually associated withvarious degrees of loss of specific cellular and tissue function.

Tumors are divided for better classification into:

I. According to their Biological Behavior:

-   -   1. Benign tumors with differentiated cells and slow, locally        displacing growth.    -   2. Malignant tumors with cell nuclear polymorphism, cell atypia,        anaplasia and infiltrating, usually rapid, destructive growth        and metastasis.    -   3. Semimalignant tumors with the histological characteristics of        malignant tumors and locally infiltrating growth, but usually        without metastasis.

II. Histogenic Classification:

-   -   This entails the tumors being classified on the basis of the        tissue from which they have originated in the developmental        history. There are:    -   1. Epithelial tumors originating from ectoderm and entoderm:        -   a) benign tumors such as, for example, adenoma, papilloma            and polyps        -   b) malignant tumors such as, for example, carcinoma.    -   2. Mesenchymal tumors originating from the mesoderm:        -   a) benign tumors, such as, for example, lipoma, fibroma,            osteoma, myoma, leiomyoma, rhabdomyoma, chondroma,        -   b) malignant tumors such as, for example, the sarcomas.    -   3. Embryonic tumors have originated from undifferentiated        tissue. These include for example nephroblastomas,        neuroblastomas, medulloblastomas, retinoblastomas and embryonic        rhabdomyosarcomas and teratomas.

III. Classification According to Clinical and Pathological Findings:

-   -   Inter alia the TNM classification, grading, Laurén        classification, Dukes classification, Kieler classification,        Rappaport classification etc apply here.

Even this brief review of tumor classification shows the diversity (andin some cases antagonism) existing within the various types of tumors.Thus, for example, a distinction is to be made not only between benignand malignant tumors but also between mortality or lethality of theindividual tumors and the probability that a benign tumor developsfurther to a malignant tumor.

Some tumors such as, for example, breast carcinomas (breast cancer), thecommonest malignant tumor of women, occur with increased frequency inparticular between the ages of 45 and 70. Early symptoms are suspiciousfindings of palpation, which are usually discovered following theexaminations for early cancer diagnosis and on regular self-examinationof the breast. Depending on the tumor stage and degree ofdifferentiation of the tumor, the prognosis in these cases may rangefrom definitely positive to very poor. As a consequence of the earlylymphogenous and hematogenous metastasis of breast carcinomas it isimportant for the tumor to be diagnosed rapidly in order to be able toinstitute therapy as early as possible.

Prostatic carcinomas (carcinoma of the prostate) is on the other handthe commonest malignant tumor in men, which occurs in particular betweenthe ages of 50 and 70. They are in most cases adenocarcinomas. Thismalignant tumor initially spreads through infiltrating growth within theprostate, and later there is infiltration of vesicular glands and pelvicconnective tissue, and relatively rarely of the rectum, bladder orurethra. Lymphogenous and/or hematogenous metastasis takes place. Thetherapy depends on the histological degree of differentiation andclinical stage and usually involves radical prostatectomy with regionallymph node extirpation, and in the advanced stage withdrawal of male sexhormones. In these cases, too, the prognosis depends on the stage of thecarcinoma. Whereas in a very early stage a radical prostatectomy isfollowed by cure in about 90% of cases, in an advanced stage theprognosis tends to be pessimistic.

Prostatic carcinomas need to be diagnostically differentiated fromhyperplasia of the prostate. Hyperplasia of the prostate is a benigntumor. In these cases there is enlargement of the prostate through anumerical increase in cells and glands of the stroma. Hyperplasia of theprostate is the commonest cause of impairments of bladder emptying inmen. Its clinical onset is in particular between the ages of 40 and 50.Progression is slow and episodic. Moreover, symptoms in most casesappear only after some years with a gradual weakening of the urinarystream and delayed start of urination. In these cases, theadministration of phytotherapeutic agents may be considered as therapyor alleviation of the symptoms.

Since early recognition, i.e. diagnosis, of tumors is generallyimportant for starting therapy quickly, and the prognosis is also betterwhen the tumor is diagnosed earlier, a number of so-called tumor markersare in clinical use. Substances and cellular changes whose qualitativeor quantitative analysis may enable statements to be made about thepresence, progression or prognosis of (malignant) disorders aregenerally referred to as tumor markers. Tumor markers are classified as:

1. Cellular Tumor Markers:

-   -   These include inter alia cell membrane-associated tumor        antigens, receptors (e.g. hormone receptors, receptors for        growth-promoting substances in leukemia) and cell markers which        indicate an increased expression of oncogenes and a monoclonal        cell growth, and molecular genetic cellular changes, especially        chromosome aberrations.

2. Humoral Tumor Markers:

-   -   These are (usually physiologically occurring) substances which        are detectable in increased concentrations, compared with        physiological conditions, in serum, urine and other body fluids        and which are synthesized and/or secreted by the tumor tissue,        released by oncolysis or formed as a response of the organism to        a tumor. The physiological significance of tumor markers is only        inadequately known. They usually have no immunogenic effect in        the human body. The clinical (diagnostic) significance depends        on their specificity and sensitivity. The humoral tumor markers        are divided into two groups. The first group comprises the        humoral tumor markers which are produced by the tumor itself.        These include, for example, tumor-associated antigens, certain        hormones (e.g. gastrin, cortisol etc), enzymes (e.g.        neuron-specific enolase (NSE)), and proteins (e.g. Bence-Jones        protein). The second group comprises the tumor markers which,        although induced by the tumor, are not produced by it. Important        humoral tumor markers of this group are, for example, alkaline        phosphatase (AP), LDH, neopterin etc.

The statements made above show how important selective and sensitivetumor detection methods are. There is in addition a great need for noveltargets for tumor therapy.

Accordingly, the object of the invention is to provide novel activeingredients and targets for diagnostic and therapeutic applications intumor therapy.

It has surprisingly been possible to show in experiments with varioustumors that certain proteins were synthesized and/or secreted only inthe tissue affected by tumors. These proteins thus play an importantrole in the development of tumors and the progression of a neoplasticdisease.

Accordingly, the object of the invention is achieved by the subjectmatter of independent claims 1 to 5, 14, 18 and 22. Preferredembodiments are specified in the dependent claims 6 to 13, 15 to 17, 19to 21 and 23. The content of all these claims is hereby included in thedescription by reference.

It is possible according to the invention to use at least one activeingredient for the prevention or treatment of tumors, in particularmalignant tumors. In this connection, this active ingredient influencesthe expression and/or the function of proteins synthesized and/orsecreted by the tumor in eukaryotic cells, whereby the increase intissue volume and/or metastasis of the tumor is at least partiallyinhibited. By influencing the expression and/or function of the proteinssynthesized and/or secreted by the tumor is meant in particularinhibition of these proteins. This active ingredient can further be usedto produce a medicament or a pharmaceutical composition for theprevention or treatment of tumors.

Also claimed is the use of a substance for detecting the expressionand/or the function of proteins synthesized and/or secreted by tumors,in particular malignant tumors, in eukaryotic cells, for diagnosingdisorders associated with these tumors. Disorders associated with thesetumors include, for example, the abovementioned prostatic carcinomas,hyperplasia of the prostate (hypertrophy of the prostate) etc. However,all other tumor-associated disorders in which the proteins of theinvention are synthesized and/or secreted are also encompassed by theinvention.

In a further preferred embodiment, a method for the prevention ortreatment of tumors, in particular malignant tumors, is claimed, whereeukaryotic cells are treated with an active ingredient which influences,in particular inhibits, the expression and/or the function of proteinssynthesized and/or secreted by tumors, and thus at least partiallyinhibits the increase in tissue volume and/or the metastasis of thetumors.

In addition, in a further preferred embodiment, a method for diagnosingdisorders associated with tumors, in particular malignant tumors, isclaimed, where eukaryotic cells are brought into contact with asubstance which detects the expression and/or the function of proteinssynthesized and/or secreted by these tumors.

The proteins synthesized and/or secreted by the tumors may be, in aparticularly preferred embodiment, the proteins listed in table I shownbelow. Thus, the substance employed for detecting and/or for diagnosingtumor-associated disorders may be for example an antibody which isdirected against these proteins and is employed in a detection methodknown to the skilled worker, such as, for example, ELISA (enzyme-linkedimmuno sorbent assay). In such so-called immunoassays, the specificantibody directed against the antigen to be determined (or in the caseof antibody determinations homologous test antigens) is bound to asupport substance (e.g. cellulose, polystyrene) on which immunecomplexes form after incubation with the sample. In a subsequent step, alabeled antibody is added to these immune complexes. It is possible, byadding a homogeneous substrate to the reaction mixture, to visualize theimmune complex-bound enzyme-substrate complexes and estimate the antigenconcentration in the sample via a photometric determination of theimmune complex-bound marker enzymes by comparison with standards ofknown enzymic activity. Further substances which can be used for thediagnostic detection are, for example, so-called oligonucleotides whichare suitable, with the aid of the so-called polymerase chain reaction(PCR), via a molecular genetic method in which there is selectiveamplification of particular DNA segments, for achieving quantitativedetection of the investigated proteins. Further methods allowing a knowntarget protein to be detected quantitatively or qualitatively arefamiliar to the skilled worker. Active ingredients which can be used forat least partial inhibition of these proteins are likewise known to theskilled worker. Thus, for example, so-called antisense sequences can beused as active ingredient. It is additionally possible to usegenetically modified mutants of these proteins according to theinvention as active ingredient, e.g. so-called deficient mutants inwhich the enzymatic activity has been eliminated. Protein NM_(—)018946,Sialic acid synthase, identified by accession number gi 12056473 andshown in row 6 of Table 1 corresponds to SEQ ID NO: 1. ProteinAB_(—)001517, KNP-I beta, identified by accession number gi_(—)2250701and shown in row 16 of Table 1 corresponds to SEQ ID NO: 2.

TABLE I Detected tissue-specific proteins MW MW Expr. Acc no ProteinScores Theo. range pI cancer 1 gi|1085373 protein disulfide-isomerase EC319 57883 60000 5.9 ++ 5.3.4.1) ER60 precursor - human 6.1 ++ 2gi|1374715 ATP synthase beta chain, mitochondrial 284 56525 55000 5.0 ++precursor 3 gi|14729950 (XM_028869) isocitrate dehydrogenase 94 4751542000 6.8 ++ 1 (NADP+), soluble [Homo sapiens] 4 gi|184326 M12387)haptoglobin precursor 100 47073 22000 5.8 + [Homo sapiens] 5 gi|4505763(NM_000291) phosphoglycerate 136 45826 40000 8.7 ++ kinase 1 [Homosapiens] 6 gi|12056473 (NM_018946) N- 167 41698 37000 6.5 ++acetylneuraminic acid phosphate synthase, sialic acid synthase, sialicacid phosphate 7 gi|13111901 (BC003119) Similar to ATP 76 40614 250007.1 ++ synthase, H+ transporting, mitochondrial F1 complex, alphasubunit, isofo 8 gi|4757756 (NM_004039) annexin A2, 81 39288 25000 7.1−− annexin II, lipocortin II, Annexin II (lipocortin I), calpactin I,heavy po 9 gi|5174541 (NM_005918) malate dehydrogenase 177 36925 320009.2 ++ 2, NAD (mitochondrial), 32000 9.5 ++ Malate dehydro-genase,mitochondrial 10 gi|4506237 (NM_002818) proteasome 96 27863 30000 5.5 o(prosome, macropain) activator 30000 5.6 ++ subunit 2 (PA28 beta),Proteasome activator s 11 gi|225915 gamma seminoprotein [Homo 120 2784325000 6.5 ++ sapiens] 25000 6.8 ++ 32000 6.8 o 32000 7.1 o 32000 7.5 o12 gi|999892 Chain A, Triosephosphate 200 27407 25000 6.5 + Isomerase(Tim) (E.C.5.3.1.1) 25000 6.8 + Complexed With 2- 25000 7.1 oPhosphoglycolic Acid 13 gi|4507359 (NM_003186) transgelin; 100 2263815000 5.3 − smooth muscle protein 20000 6.6 − 22-alpha; 22 kDaactin-binding 20000 6.1 −− protein; SM22-alpha 21000 6.6 −− 23000 6.9 o23000 8.2 o 23000 9.1 o 23000 9.6 + 14 gi|5729842 (NM_006708) glyoxalaseI, 135 21415 22000 4.8 ++ lactoyl glutathione lyase, lactoylglutathione22000 4.9 ++ lyase [Homo 22000 5.0 ++ sapiens] 15 gi|4505621 (NM_002567)prostatic binding 160 21398 20000 7.9 + protein,phosphatidylethanolamine binding protein [Homo sapiens] 16 gi|2250701(AB001517) KNP-I beta protein 68 20819 25000 7.1 ++ [Homo sapiens] 17gi|4827038 (NM_005079) tumor protein 131 19851 25000 4.7 ++ D52 [Homosapiens] 18 gi|5031635 (NM_005507) cofilin 1 (non- 125 19199 17000 8.5 omuscle) [Homo sapiens] 18000 6.5 ++ 19 gi|4507387 (NM_003197)transcription 74 19177 18000 4.2 − elongation factor B polypeptide1-like, organ of Corti protein 2 [Homo sapiens] 20 gi|4503545(NM_001970) eukaryotic 102 17530 17000 5.1 + translation initiationfactor 5A [Homo sapiens] 21 gi|10120703 Chain A, Structure of Human 16413930 17000 5.6 ++ Transthyretin Complexed With 37000 5.6 oBromophenols: A New Mode Of Binding 22 gi|4557581 Fatty acid bindingprotein 5 60* 15155 18000 6.5 ++ (psoriasis-associated) Scores = hitsfound with the aid of the MASCOT technique MW theo. = theoretical(calculated) molecular weight MW range = found in the molecular weightrange of the indicated marker proteins pI = isoelectric point Expr.cancer (o = found both in malignant and benign tissue; + upregulated; −downregulated).

In a further preferred embodiment, the active ingredient or thesubstance is directed against the proteins themselves which aresynthesized and/or secreted by the tumors. As already described, theactive ingredients may be for example antisense sequences. These arethen directed directly against the synthesized and/or secreted proteins.The active ingredient may additionally constitute genetically modifiedmutants. Thus, it is possible to construct, for example, by geneticengineering methods, mutants in which the catalytic center is eliminated(so-called deficient mutants). In these cases, although thetumor-associated proteins are synthesized, they have no or only areduced enzymatic activity. These deficient mutants which havepreviously been inserted into the tumor tissue are unable to comply withthe task assigned to them in the tumor tissue, by which means theincrease in tissue volume and/or the metastasis of the tumor is at leastpartially inhibited.

In another preferred embodiment of the invention, the active ingredientis directed against activators, inhibitors, regulators and/or biologicalprecursors of proteins synthesized and/or secreted by tumors. Theseactivators, inhibitors, regulators and/or biological precursors may befor example members, located up- and downstream, of the transductioncascade of the proteins listed in table I, transcription factors whichregulate the level of expression of said proteins, but also previouslyunknown molecules which are influenced by the active ingredient and areinvolved in the expression and/or function of said proteins.

It is possible in the invention to use both known and unknown activeingredients or substances. Thus, for example, the active ingredient orthe substance may be a polynucleotide which encodes a peptide, inparticular a polypeptide, this peptide preferably influencing, inparticular inhibiting, the expression and/or function of proteinssynthesized and/or secreted by tumors. The active ingredient or thesubstance may further be a peptide, preferably a polypeptide, thispeptide preferably influencing, in particular inhibiting, the expressionand/or function of proteins synthesized and/or secreted by tumors. Theactive ingredient or the substance may further be a small molecularcompound, preferably a small molecular compound having a molecularweight (MW) of <1000.

In a particularly preferred embodiment, the malignant tumor is aprostatic carcinoma. As already described, prostatic carcinomasrepresent the commonest malignant tumors in men. Only if a prostatictumor can be detected in an early stage e.g. by prostate-specificantigen-based mass screenings (Bartsch G, Horninger W, Klocker H,Reissigl A, Oberaigner W, Schonitzer D, Severi G, Robertson C, Boyle P:Prostate cancer mortality after introduction of prostate-specificantigen mass screening in the Federal State of Tyrol, Austria. Urology2001; 58:417-24) is it possible to consider the pure (preventive)surgical removal of the prostate (Bukkapatnam R, Pow-Sang J M: Radicalprostatectomy in the management of clinically localized prostate cancer.Cancer Control 2001; 8:496-502; Pentyala S N, Lee J, Hsieh K, Waltzer WC, Trocchia A, Musacchia L, Rebecchi M J, Khan S A: Prostate cancer: acomprehensive review. Med Oncol 2000; 17:85-105). For the advanceddisorder which is no longer limited to one organ, preventive removal ofthe prostate is no longer adequate. A possible choice of therapy forthese prostatic tumors (prostatic carcinomas), some of which areinoperative, is, as already described, inhibition of the male sexhormones (Hussain A, Dawson N: Management of advanced/metastaticprostate cancer: 2000 update. Oncology (Huntingt) 2000; 14; 1677-88;discussion 1688, 1691-4). This inhibition of the production of male sexhormones, in some cases in combination with surgical or pharmacologicalcastration, partly inhibits the proliferation and metastasis of thetumor and thus permits it to be controlled for a certain period (Afrin LB, Ergul S M: Medical therapy of prostate cancer: 1999. J S C Med Assoc2000; 96:77-84; Auclerc G, Antoine E C, Cajfinger F, Brunet-Pommeyrol A,Agazia C, Khayat D: Management of advanced prostate cancer. Oncologist2000; 5:36-44). Most prostatic tumors develop over time a certainresistance to this endocrinological therapy and, over time, they becomeandrogen-insensitive (Eder I E, Culig Z, Putz T, Nessler-Menardi C,Bartsch G, Klocker H: Molecular biology of the androgen receptor: frommolecular understanding to the clinic. Eur Urol 2001; 40:241-51;Crawford E D, Rosenblum M, Ziada A M, Lange P H: Hormone refractoryprostate cancer. Urology 1999; 54:1-7). Further possible therapeuticoptions such as, for example the use of cytotoxic agents (Heidenreich A,von Knobloch R, Hofmann R: Current status of cytotoxic chemotherapy inhormone refractory prostate cancer: Eur Urol 2001; 39:121-30), genetherapy (Miyake H, Hara I, Kamidono S, Gleave M E: Novel therapeuticstrategy for advanced prostate cancer using antisenseoligodeoxynucleotides targeting anti-apoptotic genes upregulated afterandrogen withdrawal to delay androgen-independent progression andenhance chemosensitivity. Int J. Urol 2001; 8:337-49) and immunotherapy(Rini B I, Small E J: Immunotherapy for prostate cancer. Curr Oncol Rep2001; 3:418-23), although undergoing clinical testing, have not to datebeen able to achieve significant success in the treatment of prostaticcarcinoma (DiPaola R S, Kumar P, Hait W N, Weiss R E: State-of-the-artprostate cancer treatment and research. A report from the CancerInstitute of New Jersey. N J Med 2001; 98:23-33). Identification ofgenes which are expressed only in tumors, or in which differentexpression can be detected in benign and malignant tumors, is thereforea promising approach to the therapy of these tumors (Magee J A, Araki T,Patil S, Ehrig T, True L, Humphrey P A, Catalona W J, Watson M A,Milbrandt J: Expression profiling reveals hepsin overexpression inprostate cancer. Cancer Res 2001; 61:5692-6; Welsh J B, Sapinoso L M, SuAl, Kern S G, Wang-Rodriguez J, Moskaluk C A, Frierson H F, Jr., HamptonG M: Analysis of gene expression identifies candidate markers andpharmacological targets in prostate cancer. Cancer Res 2001; 61:5974-8;Stamey T A, Warrington J A, Caldwell M C, Chen Z, Fan Z, Mahadevappa M,McNeal J E, Nolley R, Zhang Z: Molecular genetic profiling of Gleasongrade 4/5 prostate cancers of compared to benign prostatic hyperplasia.J Urol 2001; 166:2171-7; Dhanasekaran S M, Barrette T R, Ghosh D, ShahR, Varambally S, Kurachi K, Pienta K J, Rubin M A, Chinnaiyan A M:Delineation of prognostic biomarkers in prostate cancer. Nature 2001;412:822-6; Waghray A, Schober M, Reroze F, Yao F, Virgin J, Chen Y Q:Identification of differentially expressed genes by serial analysis ofgene expression in human prostate cancer. Cancer Res 2001; 61:4283-6;Chaib H, Cockrell E K, Rubin M A, Macoska J A: Profiling andverification of gene expression patterns in normal and malignant humanprostate tissues by cDNA microarray analysis. Neoplasia 2001; 3:43-52;Chetcuti A, Margan S, Mann S, Russell P, Handelsman D, Rogers J, Dong Q:Identification of differentially expressed genes in organ-confinedprostate cancer by gene expression array. Prostate 2001; 47:132-40). Itis therefore possible according to the invention to offer a therapeuticapproach to the treatment of these tumors through inhibition of thedescribed tumor-associated proteins.

The active ingredient or the substance can moreover be administeredorally, intravenously, topically and/or by inhalation in a furtherpreferred embodiment. The administration form depends on the tumoritself and on the patient's constitution. Further administration formsare known to the skilled worker.

The invention additionally includes a pharmaceutical composition whichcomprises an effective amount of at least one active ingredient whichinfluences, in particular inhibits, the expression and/or the functionof proteins synthesized and/or secreted by tumors, in particular bymalignant tumors, and where appropriate a pharmaceutical carrier. Theactive ingredient may be a polynucleotide which encodes a peptide, inparticular a polypeptide, this peptide preferably influencing, inparticular inhibiting, the expression and/or function of proteinssynthesized and/or secreted by tumors, in particular malignant tumors.The active ingredient may further be a peptide, preferably apolypeptide, this peptide preferably influencing, in particularinhibiting, the expression and/or function of proteins synthesizedand/or secreted by tumors, in particular malignant tumors. The activeingredient may also be a so-called small molecular compound, preferablya small molecular compound having a molecular weight (MW) of <1000. Forthe further features of such an active ingredient, reference is made tothe corresponding previous text of the description.

The invention also encompasses a pharmaceutical composition whichcomprises an effective amount of at least one active ingredient whichinfluences, in particular inhibits, the expression and/or function ofactivators, inhibitors, regulators and/or biological precursors ofproteins synthesized and/or secreted by tumors, in particular malignanttumors, and where appropriate a pharmaceutical carrier. The activeingredient in this case may be a polynucleotide which encodes a peptide,preferably a polypeptide, this peptide preferably influencing, inparticular inhibiting, the expression and/or function of activators,inhibitors, regulators and/or biological precursors of proteinssynthesized and/or secreted by tumors, in particular malignant tumors.The active ingredient may also be a peptide, preferably a polypeptide,this peptide preferably influencing, in particular inhibiting, theexpression and/or function of activators, inhibitors, regulators and/orbiological precursors of proteins synthesized and/or secreted by tumors,in particular malignant tumors. A further active ingredient of theinvention may be for example a small molecular compound, preferably asmall molecular compound having a molecular weight (MW) of <1000. Forthe further features of such an active ingredient, and of theactivators, inhibitors, regulators and/or biological precursors againstwhich this active ingredient is directed, reference is made to thecorresponding previous text of the description.

Finally, the invention includes a diagnostic kit, this diagnostic kitcomprising at least one substance for detecting the expression and/orfunction of proteins synthesized and/or secreted by tumors, inparticular malignant tumors, for diagnosing disorders associated withthese tumors. It is moreover possible for example for a correspondingdisorder to be a prostatic carcinoma, which is claimed in a particularlypreferred embodiment. For further features of such a substance,reference is made to the corresponding previous text of the description.

The existing features and further features of the invention are evidentfrom the following description of preferred embodiments in conjunctionwith the dependent claims and the figures. It is possible in thisconnection for the individual features to be implemented each on its ownor in a combination of a plurality with one another.

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

The figures show:

FIG. 1: Diagrammatic representation of the experimental design

FIG. 2: Gel electrophoretic and MS analysis of benign and malignantprostatic tissue and the equivalent spots.

FIG. 3: Preparative gels with tissue-specific protein expression.

MATERIAL AND METHODS Patients and Tissue Samples

Benign and malignant prostatic tissue were obtained from patientspreviously subjected to a prostatectomy. The patients had beenidentified with the aid of PSA (prostata specific antigen) screening,and the tumors were confirmed with ultrasound. Consent was obtained fromeach patient before the operation was performed.

Immediately after the prostate was removed it was transferred into asterile box and cooled therein. The samples were transferred to thepathologist, where tissue sections 0.5 to 1 cm thick were made. Thesections were divided into a left and a right half, embedded in afreezing matrix and shock-frozen. The remainder of the prostate wasfixed in formalin and treated further in accordance with standardmethods. To obtain tissue samples, thin sections were taken from bothsides of the prostate and stained with hematoxilin-Eosin. Thepathologist located and marked the tumor. Tumor tissue was taken fromthe hematoxilin-Eosin-stained strips and stored at −80° C. Marked benigncontrol strips were taken from regions not affected by tumor andsubjected to an identical treatment. The tumors were stored at −80° C.where necessary.

Proteomics Analysis:

Protein alkylation, iodination, 2D-PAGE and the data analysis werecarried out in accordance with standard methods. Radioactive iodineoriginates from Amersham Biosciences (Freiburg). Proteins were labeledwith iodine I-125 or I-131 singly with identical concentrations ofiodine. All radioactive operations were generated in accordance with the“Radiation protection regulations 2001” (Germany). For the 2D PAGE, thesamples were mixed together and fractionated by co-electrophoresis inaccordance with the diagram of FIG. 1. The radioactive measurements werecarried out with a multiple photon detection (MPD) or a Phosphorimager(Fuji FLA 3000, Raytest, Straubenhard, Germany).

The multiple photon detection measurements were carried out on a 1600pixel MPD imager (BioTracers Inc., Herndon, USA) in accordance with themanufacturer's instructions for at least 24 hours, for a 24 cm×24 cmregion per measurement. The scanning in these cases was set at 0.5 mmper pixel. In some cases, small regions were scanned for a longer timein order to achieve a higher sensitivity, or scanning was at 0.25 mm perpixel in order to increase the resolution. The MPD imagers were moreoverset to detect either I-125 or I-131 in each measurement. Because of theshorter half-life of I-131, this isotope was always measured beforeI-125 for each individual sample. MPD data were converted into matrixdata using the IMAGEVIEW software (BioTraces). These data were thenanalyzed using the BIOPREPARATION software in accordance with themanufacturer's instructions. Radioactive data were converted into TIFFdata with the aid of an algorithm for analysis of conventional software.Some radioactive measurements were carried out with a Phosphorimager.

Protein identification was carried out using preparative 2D PAGE gels.These gels contain up to 1 mg of protein which have been iodinated underidentical chemical conditions as for the radioactive iodination, butwith addition of non-radioactive iodine molecules. Silver-stainedproteins with a migration behavior like the radioactive proteins werecollected automatically with the aid of the Genomic Solutions Flexysrobot. In some cases, the silver-stained proteins were removed at theplaces on the gel where no radioactive signal was present but where thesilver gels differed between benign and malignant tissue. Gel portionswere digested automatically with trypsin in a Genomic SolutionsInvestigator Progest and 10% of the resulting proteins were loaded ontoa MALDI template with the aid of a Genomic Solutions Investigator ProMSrobot. MALDI-TOF was carried out in a Bruker AutoFlex in accordance withthe manufacturer's information. Where necessary, up to 90% of thepeptide obtained by trypsin digestion was analyzed with the aid of theLC/MS/MS method. In this case, an LC-Packing Ultimate micro HPLC wasconnected to a Bruker Esquire ion trap mass spectrometer, or the sampleswere analyzed with the aid of the nanospray MS/MS method on the samemass spectrometer. Protein identification was carried out with the aidof the MASCOT program of version 1.07 (Matrix Science, UK) using our ownalgorithms.

Results:

FIG. 1 shows a diagrammatic representation of the experimental design onwhich the results are based. This entails two samples A and B to beanalyzed each being labeled separately with I-125 and I-131 in order toobtain the iodinated samples 125-A, 125-B, 131-A and 131-B. Each sampleis analyzed in gels 1 and 2 in each case comparing with the same butdifferently labeled sample, as also depicted in the figures. Samples Aand B are analyzed in gels 3 and 4, comparing with the correspondingother sample under the same conditions. These gels therefore containfour replicates of samples A and B, these being directly related tosamples A and B.

FIG. 2 shows the results of these experiments. FIG. 2A thereinrepresents malignant, 2 B benign and 2 C the comparative gel (2 A+2 B).FIG. 2C shows the different expression of individual proteins in thedifferent tissues. The results of these analyses are compiled in table Iwhich has already been shown.

FIG. 3 shows the proteins found for the example of two preparative gels(pH range 4-10). It should be pointed out in particular that severalisoforms of individual proteins were found. Thus, for example, a totalof 5 isoforms which were detected at pH 6.5-7.5 exist for protein 11(gamma-seminoprotein), thus demonstrating the existence of severalisoforms of one and the same protein. It was additionally possible toshow that there is formation not only of particular proteins but inparticular also of specific isoforms of individual proteins in tumors.It is also possible to show that in some cases the expression ofparticular proteins (see table I) is inhibited, i.e. downregulated.These proteins thus represent suitable targets for known and yet to bedeveloped active ingredients for the treatment of the disordersassociated therewith, and suitable targets for detecting thesedisorders.

1. A method for diagnosing prostatic carcinomas in a patient, comprisingcontacting prostate tissue from said patient with an antibody which isdirected against proteins synthesized and/or secreted by carcinomas,wherein the proteins are selected from the group consisting of sialicacid synthase (SEQ ID NO:1) and KNP-I beta (SEQ ID NO:2), and wherein ahigher level of said proteins in said prostate tissue, as compared tothe level of said proteins in normal prostate tissue, indicates saidpatient has a prostatic carcinoma.